Cell line

ABSTRACT

The invention provides a cell line complementing HSV ICP4, ICP27, and UL55 genes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 11/694,118, filed Mar. 30, 2007, which is a divisional ofco-pending U.S. patent application Ser. No. 11/261,389, which was filedon Oct. 28, 2005, claiming the benefit of U.S. Provisional PatentApplication 60/622,889, filed Oct. 28, 2004. U.S. patent applicationSer. No. 11/694,118 also is a continuation of patent applicationPCT/US2005/39162, which was filed on Oct. 28, 2005, designating theUntied States and also claiming the benefit of U.S. Provisional PatentApplication 60/622,889, filed Oct. 28, 2004. The contents of each ofthese applications are incorporated herein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant NumbersNS044507, NS38850 and NS43247, awarded by the United States NationalInstitute of Neurological Disorders. The Government has certain rightsin the invention.

BRIEF SUMMARY OF THE INVENTION

The invention provides a cell line complementing HSV ICP4, ICP27, andUL55 genes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of exemplary vector constructs thatcan be used in the invention.

FIG. 2 is a representation of QHGAD67 transduction by footpadinoculation increased GAD67 mRNA in lumbar dorsal root ganglia (DRG).One week after subcutaneous inoculation of 30 μl of 1×10⁹ pfu/ml QHGAD67or Q0ZHG into one hind paw total RNA was extracted from the pooled L4-L6DRG (500 ng), amplified by real-time PCR, and quantitated using GAPDH asa standard. The amount relative to Q0ZHG-transduced ganglia isrepresented. Means±Standard Error Mean (SEM), N=6.

FIG. 3 represents protein from the dorsal quadrant of lumbar spinal corddetermined by Western blot using β-actin as an internal standard andquantitated by relative optical density. Means±SEM, N=6, *P<0.05.increased GAD67-like immunoreactivity after transduction with QHGAD67.

FIG. 4A is a representation of the amount of gamma amino butyric acid(GABA) released from primary DRG neurons transduced in vitro at anm.o.i. of 1 increased substantially in QHGAD67-infected compared tocontrol or Q0ZHG-infected cells. GABA released over 5 min was determinedby HPLC as described under Materials and Methods. The measurement ofGABA concentration/well was performed three times and triplicate sampleswere used for each condition. Means±SEM, *P<0.01 vs. Q0HG or vehicle.FIG. 4B is a representation of the amount of GABA released from nerveterminals in spinal cord in vivo was determined by HPLC inmicrodialysate of dorsal horn. One week after subcutaneous inoculationof 30 μl of 1×10⁹ pfu/ml QHGAD67 into one hind paw the amount of GABA(pmol/10 μl fraction of microdialysate) was substantially increased inQHGAD67-inoculated compared to control animals. Means±SEM, N=6, *P<0.05.

FIGS. 5A-D are representations of the mechanical allodynia and thermalhyperalgesia were significantly reduced by QHGAD67 inoculation. FIGS. 5Aand 5B demonstrate that one week after hemisection there was a decreasein paw withdrawal threshold (mechanical allodynia), which persisted for15 weeks as shown in vehicle-treated animals (x). Inoculation withQHGAD67 produced an antialiodynic effect reflected in an increasedthreshold value (open circle). Seven weeks after initial inoculation theantialiodnyic effect of QHGAD67 decreased, but reinoculation of QHGAD67into the same animals reestablished the antinociceptive effect (*P<0.05,**P<0.01 vs. Q0ZHG-inoculated, N=6). (A) Ipsilateral and (B)contralateral to hemisection. FIGS. 5C and 5D demonstrate that one weekafter hemisection there was a significant decrease in paw withdrawallatency (thermal hyperalgesia) and injection of the vector resulted in asignificant increase in paw withdrawal latency (open circles). By 7weeks after initial inoculation both the antihyperalgesic effects ofQHGAD67 had decreased, but reinoculation of QHGAD67 reestablished theantihyperalgesic effects (*P<0.05, **P<0.01 vs. Q0ZHG-inoculated, N=6.(C) Ipsilateral and (D) contralateral to hemisection. Q0ZHG-inoculatedanimals (open triangles) were indistinguishable from vehicle-treatedcontrols in all cases (A-D).

FIGS. 6A and 6B demonstrate that bicuculline (0.5 μg) or phaclofen (0.8μg) administrated intrathecally 3 weeks after hemisection and 2 weeksafter footpad inoculation partially reversed the (A) antialiodynic and(B) antihyperalgesic effects of vector inoculation. The dotted linerepresents the mean threshold (A) and latency (B) in animals after SCIinoculated with control vector or vehicle. Means±SEM, N=6, *P<0.05,**P<0.01 vs vehicle-treated.

FIG. 7 is a histogram of the relative optical density measurements ofCGRP-like immunoreactivity in dorsal horn. The relative optical densitymeasurements were taken from a series of six continuous sections in theL5 segment of each animal. The dotted line indicates that the density ofCGRP-IR in normal spinal cord is increased substantially bothipsilateral and contralateral to T13 hemisection in animals inoculatedwith vehicle or Q0ZHG, and inoculation with QHGAD67 significantlyattenuates this increase. Means±SEM, n=6, *P<0.051 compared to vehicleor Q0ZHG.

FIG. 8 depicts SEQ ID NO:1 discussed herein.

FIG. 9 depicts SEQ ID NO:2 discussed herein.

FIG. 10 depicts SEQ ID NOs:3-8 discussed herein.

FIGS. 11( a) and 11(b) depict data demonstrating Antinociceptive effectof QOGAD67 in neuropathic pain. (A) L5 spinal nerve ligation (SNL)caused a significant decrease in the threshold to tactile stimulation,which persisted for more than 4 months. Subcutaneous inoculation ofQHGAD67 (arrow) produced an antiallodynic effect reflected in anincrease in the mechanical threshold. Reinoculation of QHGAD67 7 weeksafter the initial inoculation (arrow) reestablished the antiallodyniceffect. Results are expressed as mean±standard error of the mean. (opencircles) QHGAD67; (closed circles) QOZHG; *p<0.05; **p<0.01; n=8 animalsper group. (B) L5 SNL also caused a significant thermal hyperalgesia,which persisted for 6 weeks. Inoculation with QHGAD67 (arrow), but notQOZHG, reversed the thermal hyperalgesia induced by spinal nerve injury.*p<0.05; **p<0.01 versus QOZHG-inoculated; n=8 animals per group. Thestatistical significances of the differences were determined by analysisof variance (StatView 5.2; SAS Institute, Cary, N.C.) corrected for thenumber of post hoc comparisons using Scheffe's F test.

FIG. 12 is a histogram depicting data concerning the effect of QHGAD67on Fos-LI in dorsal horn. Fos-LI in dorsal horn induced by 10 minutes ofgentle tactile stimulation was markedly increased in rats inoculatedwith QOZHG 1 week after spinal nerve ligation (SNL) and tested 2 weekslater (3 weeks after SNL). This increase was blocked in rats with SNLthat had been inoculated with QHGAD67 1 week after SNL and tested 2weeks later (3 weeks after SNL), and it was found in laminae I-VI ofdorsal horn. Results are expressed as mean±standard error of the mean.**p<0.01; n=5 animals per group. The difference between sham-operatedand SNL animals inoculated with QOZHG was also statistically significant(p<0.01).

FIG. 13 graphically depicts data concerning the effect of QHGAD67 on thephosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2)expression in dorsal horn. Results are expressed as mean±standard errorof the mean. **p<0.01; ***p<0.001; n=5 animals per group.

FIG. 14 depicts the construction of an HSV vector having extendeddeletions of the ICP4 and ICP27 loci and a deletion of UL55.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the invention provides a vector comprising apolynucleotide sequence encoding a glutamic acid decarboxylase protein.The vector can be any suitable gene transfer vector. Examples ofsuitable vectors include plasmids, liposomes, molecular conjugates(e.g., transferrin), and viruses. Preferably, the vector is a viralvector. Suitable viral vectors include, for example, retroviral vectors,herpes virus based vectors and parvovirus based vectors (e.g.,adeno-associated virus (AAV) based vectors, AAV-adenoviral chimericvectors, and adenovirus-based vectors). One of ordinary skill in the arthas the requisite understanding to determine the appropriate vector fora particular situation.

In a preferred embodiment, the vector is a herpesviral based vector,such as based on HSV. An HSV based viral vector is suitable for use as avector to introduce a nucleic acid sequence into numerous cell types.The mature HSV virion consists of an enveloped icosahedral capsid with aviral genome consisting of a linear double-stranded DNA molecule that is152 kb. In a preferred embodiment, the HSV based viral vector isdeficient in at least one essential HSV gene. Of course, the vector canalternatively or in addition be deleted for non-essential genes.Preferably, the RSV based viral vector that is deficient in at least oneessential HSV gene is replication deficient. Most replication deficientHSV vectors contain a deletion to remove one or more intermediate-early,early, or late HSV genes to prevent replication. For example, the HSVvector may be deficient in an immediate early gene selected from thegroup consisting of: ICP4, ICP22, ICP27, ICP47, and a combinationthereof. Advantages of the HSV vector are its ability to enter a latentstage that can result in long-term DNA expression and its large viralDNA genome that can accommodate exogenous DNA inserts of up to 25 kb.HSV-based vectors are described in, for example, U.S. Pat. Nos.5,837,532, 5,846,782, and 5,804,413, and International PatentApplications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583,which are incorporated herein by reference. Preferably, the HSV vectoris “multiply deficient,” meaning that the HSV vector is deficient inmore than one gene function required for viral replication. The sequenceof HSV is available on the internet atwww.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=9629378&dopt=GenBank&term=hsv-1&qty=1, which may facilitate the generation ofdesired mutations in designing vectors.

The HSV vector can be deficient in replication-essential gene functionsof only the early regions of the HSV genome, only the immediate-earlyregions of the HSV genome, only the late regions of the HSV genome, orboth the early and late regions of the HSV genome. The HSV vector alsocan have essentially the entire HSV genome removed, in which case it ispreferred that at least either the viral inverted terminal repeats(ITRs) and one or more promoters or the viral ITRs and a packagingsignal are left intact (i.e. an HSV amplicon). The larger the region ofthe HSV genome that is removed, the larger the piece of exogenousnucleic acid sequence that can be inserted into the genome. However, itis preferred that the vector of the present invention be a non-ampliconHSV vector.

It should be appreciated that the deletion of different regions of theHSV vector can alter the immune response of the mammal. In particular,the deletion of different regions can reduce the inflammatory responsegenerated by the HSV vector. Furthermore, the HSV vector's protein coatcan be modified so as to decrease the HSV vector's ability or inabilityto be recognized by a neutralizing antibody directed against thewild-type protein coat.

The HSV vector, when multiply replication deficient, preferably includesa spacer element to provide viral growth in a complementing cell linesimilar to that achieved by singly replication deficient HSV vectors.The spacer element can contain any nucleic acid sequence or sequenceswhich are of the desired length. The spacer element sequence can becoding or non-coding and native or non-native with respect to the HSVgenome, but does not restore the replication essential function(s) tothe deficient region. In addition, the inclusion of a spacer element inany or all of the deficient HSV regions will decrease the capacity ofthe HSV vector for large inserts. The production of HSV vectors involvesusing standard molecular biological techniques well known in the art.

Replication deficient HSV vectors are typically produced incomplementing cell lines that provide gene functions not present in thereplication deficient HSV vectors, but required for viral propagation,at appropriate levels in order to generate high titers of viral vectorstock. A preferred cell line complements for at least one and preferablyall replication essential gene functions not present in a replicationdeficient HSV vector. The cell line also can complement non-essentialgenes that, when missing, reduce growth or replication efficiency (e.g.,UL55). The complementing cell line can complement for a deficiency in atleast one replication essential gene function encoded by the earlyregions, immediate-early regions, late regions, viral packaging regions,virus-associated regions, or combinations thereof, including all HSVfunctions (e.g., to enable propagation of HSV amplicons, which compriseminimal HSV sequences, such as only inverted terminal repeats and thepackaging signal or only ITRs and an HSV promoter). The cell linepreferably is further characterized in that it contains thecomplementing genes in a non-overlapping fashion with the HSV vector,which minimizes, and practically eliminates, the possibility of the HSVvector genome recombining with the cellular DNA. Accordingly, thepresence of replication competent HSV is minimized, if not avoided inthe vector stock, which, therefore, is suitable for certain therapeuticpurposes, especially gene therapy purposes. The construction ofcomplementing cell lines involves standard molecular biology and cellculture techniques well known in the art.

When the vector is a replication deficient HSV, the nucleic acidsequence encoding the protein (e.g., GAD protein) is preferably locatedin the locus of an essential HSV gene, most preferably either the ICP4or the ICP27 gene locus of the HSV genome. The insertion of a nucleicacid sequence into the HSV genome (e.g., the ICP4 or the ICP27 genelocus of the HSV genome) can be facilitated by known methods, forexample, by the introduction of a unique restriction site at a givenposition of the HSV genome.

A preferred HSV vector for use in the context of the invention containsexpanded ICP4, or ICP27 deletions, and preferably both. By “expanded”deletions in this context, it is meant that the preferred vectors haveno homologous sequences at either or both of these loci relative to thecomplementing cell line used for their production. Desirably, the virushas no remaining ICP4 or ICP27 (or both) coding or promoter sequences.Preferably, the deletion in ICP27 extends as well into the UL55 locus,and desirably both genes are deleted. Thus, a most preferred virus foruse in the invention contains extended deletions in ICP4, ICP27 and UL55 such that there is no viral homology to these genes used in acomplementing cell line. Desirably, the vector further does not includeany homologous DNA sequences to that employed in the complementing cellline (e.g., even using different regulatory sequences andpolyadenylation sequences).

As noted above, cell lines complementing the function of genes,particularly essential genes, deleted from an HSV vector are desirable(and in the case of essential HSV genes, necessary) to replicate thevector. Thus, to employ a preferred vector that lacks both ICP4 andICP27, a cell line engineered to complement both essential genes shouldbe employed. Moreover, as UL55⁻ HSV strains grow poorly, a cell linecomplementing it is desirable for use when it is deleted from the vectorbackbone. Methods for generating complementing cell lines are known tothose of ordinary skill in the art.

As noted above, the inventive vector also comprises a nucleic acidsequence encoding a GAD protein (i.e., one or more nucleic acidsequences encoding one or more GAD proteins). The nucleic acid sequenceencoding the GAD protein can be obtained from any source, e.g., isolatedfrom nature, synthetically generated, isolated from a geneticallyengineered organism, and the like. An ordinarily skilled artisan willappreciate that any type of nucleic acid sequence (e.g., DNA, RNA, andcDNA) that can be inserted into a vector can be used in connection withthe invention.

The nucleic acid sequence of the inventive vector can encode a secretedprotein, e.g., a protein that is naturally secreted by the infectedcell. Alternatively, the nucleic acid sequence can encode a protein,such as GAD, that generates a secreted product (e.g., GABA) or peptideby enzymatic catalysis within the cell. Alternatively, the nucleic acidsequence can encode a protein that is not naturally secreted by the cell(i.e., a non-secretable protein), but which comprises a signal peptidethat facilitates protein secretion. In this manner, for example, thenucleic acid sequence encodes an endoplasmic reticulum (ER) localizationsignal peptide and the non-secretable protein. The ER localizationsignal peptide functions to direct DNA, RNA, and/or a protein to themembrane of the endoplasmic reticulum, wherein a protein is expressedand targeted for secretion. The ER localization signal peptide desirablyfunctions to increase the secretion (i.e., the secretion potential) by acell of (i) proteins that are not normally secreted (i.e., secretable)by the cell and/or (ii) proteins that are normally secreted by a cell,but in low (i.e., less than desired) quantities. The ER localizationsignal peptide encoded by the polynucleotide can be any suitable ERlocalization signal peptide or polypeptide (i.e., protein). For example,the ER localization signal peptide encoded by the nucleic acid sequencecan be a peptide or polypeptide (i.e., protein) selected from the groupconsisting of nerve growth factor (NGF), immunoglobulin (Ig) (e.g., anIg a chain leader sequence), and midkine (K), or a portion thereof.Suitable ER localization signal peptides also include those described inLadunga, Current Opinions in Biotechnology, 11, 13-18 (2000).

Although the nucleic acid sequence can encode any protein, the proteinpreferably is a GAD protein or an enkephalin. There are several isoformsof mammalian GAD encoded by several different genes, in particular,GAD25, GAD65, and GAD67. GAD65, targeted principally to membranes andnerve terminals, is regulated by pyridoxal-5′-phosphate and othercofactors. GAD65 is thought to be responsible for the packaging of GABAinto vesicles in preparation of synaptic release. Another isoform ofmammalian GAD, GAD67, is predominantly cytosolic and its enzymaticactivity appears to be regulated by protein level. GAD25 is an alternatesplicing variant of GAD67. The vector preferably comprises a nucleicacid sequence coding for GAD67. The coding sequence of the human GAD67gene and the amino acid sequence of the encoded gene product (i.e., theencoded protein) are publicly available at the National Center forBiotechnology Information (NCBI) website as GenBank Accession No. NM000817 (SEQ ID NO: 1) and NP_(—)000808 (SEQ ID NO: 2), respectively.Similarly, the coding sequence of the human enkephalin gene and theamino acid sequence of the encoded gene product (i.e., the encodedprotein) are publicly available as GenBank Accession No. NM_(—)006211.

The nucleic acid sequence can encode any variant, homolog, or functionalportion of the aforementioned proteins. A variant of the protein caninclude one or more mutations (e.g., point mutations, deletions,insertions, etc.) from a corresponding naturally occurring protein. By“naturally occurring” is meant that the protein can be found in natureand has not been synthetically modified. Thus, where mutations areintroduced in the nucleic acid sequence encoding the protein, suchmutations desirably will effect a substitution in the encoded proteinwhereby codons encoding positively-charged residues (H, K, and R) aresubstituted with codons encoding positively-charged residues, codonsencoding negatively-charged residues (D and E) are substituted withcodons encoding negatively-charged residues, codons encoding neutralpolar residues (C, G, N, Q, S, T, and Y) are substituted with codonsencoding neutral polar residues, and codons encoding neutral non-polarresidues (A, F, I, L, M, P, V, and W) are substituted with codonsencoding neutral non-polar residues. In addition, a homolog of theprotein can be any peptide, polypeptide, or portion thereof, that ismore than about 70% identical (preferably more than about 80% identical,more preferably more than about 90% identical, and most preferably morethan about 95% identical) to the protein at the amino acid level. Thedegree of amino acid identity can be determined using any method knownin the art, such as the BLAST sequence database. A “functional portion”is any portion of a GAD protein that retains the biological activity ofthe naturally occurring, full-length GAD protein at measurable levels. Afunctional portion of the GAD protein produced by expression of thenucleic acid sequence of the vector can be identified using standardmolecular biology and cell culture techniques, such as assaying thebiological activity of the GAD protein portion in human cellstransiently transfected with a nucleic acid sequence encoding the GADprotein portion.

The expression of the nucleic acid sequence encoding the protein iscontrolled by a suitable expression control sequence operably linked tothe nucleic acid sequence. An “expression control sequence” is anynucleic acid sequence that promotes, enhances, or controls expression(typically and preferably transcription) of another nucleic acidsequence. Suitable expression control sequences include constitutivepromoters, inducible promoters, repressible promoters, and enhancers.The nucleic acid sequence encoding the protein in the vector can beregulated by its endogenous promoter or, preferably, by a non-nativepromoter sequence. Examples of suitable non-native promoters include thehuman cytomegalovirus (HCMV) promoters, such as the HCMV immediate-earlypromoter (HCMV IEp), promoters derived from human immunodeficiency virus(HIV), such as the HIV long terminal repeat promoter, thephosphoglycerate kinase (PGK) promoter, Rous sarcoma virus (RSV)promoters, such as the RSV long terminal repeat, mouse mammary tumorvirus (MMTV) promoters, the Lap2 promoter, or the herpes thymidinekinase promoter (Wagner et al., Proc. Natl. Acad. Sci., 78, 1444-1445(1981)), promoters derived from SV40 or Epstein Barr virus, and thelike. In a preferred embodiment, the promoter is HCMV IEp. The HCMV IEppromoter can be inserted into the ICP4 locus of the recombinant HSV.Alternatively, expression of the nucleic acid sequence encoding theprotein can be controlled by a chimeric promoter sequence. A promotersequence is “chimeric” if it comprises at least two nucleic acidsequence portions obtained from, derived from, or based upon at leasttwo different sources (e.g., two different regions of an organism'sgenome, two different organisms, or an organism combined with asynthetic sequence). Techniques for operably linking sequences togetherare well known in the art.

The promoter can be an inducible promoter, i.e., a promoter that is up-and/or down-regulated in response to an appropriate signal. For example,an expression control sequence up-regulated by a pharmaceutical agent isparticularly useful in pain management applications. For example, thepromoter can be a pharmaceutically-inducible promoter (e.g., responsiveto tetracycline). Examples of such promoters are marketed by Ariad. Thepromoter can be introduced into the genome of the vector by methodsknown in the art, for example, by the introduction of a uniquerestriction site at a given region of the genome. Alternatively, thepromoter can be inserted as part of the expression cassette comprisingthe nucleic acid sequence coding for the protein, such as GAD. In apreferred embodiment, the inducible promoter is operably linked to thepolynucleotide sequence encoding for the GAD protein.

Preferably, the nucleic acid sequence encoding the protein furthercomprises a transcription-terminating region such as a polyadenylationsequence located 3′ of the region encoding the protein. Any suitablepolyadenylation sequence can be used, including a synthetic optimizedsequence, as well as the polyadenylation sequence of BGH (Bovine GrowthHormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein BarrVirus), and the papillomaviruses, including human papillomaviruses andBPV (Bovine Papilloma Virus).

In addition to the nucleic acid encoding the protein (including thepromoter and transcription-terminating region), the vector can compriseat least one additional nucleic acid sequence encoding at least oneother gene product, e.g., which itself performs a prophylactic ortherapeutic function, or augments or enhances a prophylactic ortherapeutic potential of the protein. The gene product encoded by theadditional nucleic acid sequence can be an RNA, peptide, or polypeptidewith a desired activity. If the additional nucleic acid sequence confersa prophylactic or therapeutic benefit, the nucleic acid sequence canexert its effect at the level of RNA or protein. Alternatively, theadditional nucleic acid sequence can encode an antisense molecule, aribozyme, a protein that affects splicing or 3′ processing (e.g.,polyadenylation), or a protein that affects the level of expression ofanother gene within the cell (i.e., where gene expression is broadlyconsidered to include all steps from initiation of transcription throughproduction of a process protein), such as by mediating an altered rateof mRNA accumulation or transport or an alteration inpost-transcriptional regulation. The additional nucleic acid sequencecan encode any one of a variety of gene products that confers aprophylactic or therapeutic benefit, depending on the intended end-useof the composition. The additional nucleic acid sequence also can encodea factor that acts upon a different target than the protein encoded bythe nucleic acid sequence of the vector, thereby providing multifactoraltreatment. The additional nucleic acid sequence can encode a chimericprotein for combination therapy. The additional gene product can besecreted, or remain within the cell in which it is produced unless oruntil the cell is lysed. A variety of gene products can enhance thetherapeutic potential of the vector.

The additional nucleic acid sequence can encode one gene product ormultiple gene products. Alternatively, multiple additional nucleic acidsequences, each encoding one or more gene products, can be inserted intothe vector. In either case, expression of the gene product(s) can beseparately regulated by individual expression control sequences, orcoordinately regulated by one common expression control sequence.Alternatively, expression of the additional nucleic acid(s) can beregulated by the same expression control sequence that regulatesexpression of the protein encoded by the nucleic acid sequence of thevector; however, any transcription terminating regions present in thenucleic acid encoding the protein would be eliminated to allow fortranscriptional read-through of the additional nucleic acid sequence(s).The additional nucleic acid sequence(s) can comprise any suitableexpression control sequence(s) and any suitabletranscription-termination region(s) discussed herein in connection withexpression of the protein produced by expression of the nucleic acidsequence of the vector.

After the vector has been created, the vector is purified. Vectorpurification to enhance the concentration of the vector in thecomposition can be accomplished by any suitable method, such as bydensity gradient purification, by chromatography techniques, or limitingdilution purification. The vector, preferably a replication deficientHSV vector, is desirably purified from cells infected with thereplication deficient HSV vector using a method that comprises lysingcells infected with the HSV vector and collecting a fraction containingthe HSV vector.

The cells can be lysed using any suitable method, such as exposure todetergents, freeze-thawing, and cell membrane rupture (e.g., via Frenchpress or microfluidization). The cell lysate then optionally can beclarified to remove large pieces of cell debris using any suitablemethod, such as gentle centrifugation, filtration, or tangential flowfiltration (TFF). The clarified cell lysate then optionally can betreated with an enzyme capable of digesting DNA and RNA (a“DNase/RNase”) to remove any DNA or RNA in the clarified cell lysate notcontained within the vector particles.

Generally, the inventive recombinant HSV is most useful when enough ofthe virus can be delivered to a cell population to ensure that the cellsare confronted with a predefined number of viruses. Thus, the presentinvention provides a stock, preferably a homogeneous stock, comprisingthe inventive HSV vector. The preparation and analysis of HSV stocks iswell known in the art. For example, a viral stock can be manufactured inroller bottles containing cells transduced with the HSV vector. Theviral stock can then be purified on a continuous nycodenze gradient, andaliquotted and stored until needed. Viral stocks vary considerably intiter, depending largely on viral genotype and the protocol and celllines used to prepare them. Preferably, such a stock has a viral titerof at least about 10⁵ plaque-forming units (pfu), such as at least about10⁶ pfu or even more preferably at least about 10⁷ pfu. In still morepreferred embodiments, the titer can be at least about 10⁸ pfu, or atleast about 10⁹ pfu, and high titer stocks of at least about 10¹⁰ pfu orat least about 10¹¹ pfu are most preferred.

The invention additionally provides a composition comprising the HSVvector and a carrier, preferably a physiologically-acceptable carrier.The carrier of the composition can be any suitable carrier for thevector. The carrier typically will be liquid, but also can be solid, ora combination of liquid and solid components. The carrier desirably is apharmaceutically acceptable (e.g., a physiologically orpharmacologically acceptable) carrier (e.g., excipient or diluent).Pharmaceutically acceptable carriers are well known and are readilyavailable. The choice of carrier will be determined, at least in part,by the particular vector and the particular method used to administerthe composition. The composition can further comprise any other suitablecomponents, especially for enhancing the stability of the compositionand/or its end-use. Accordingly, there is a wide variety of suitableformulations of the composition of the invention. The followingformulations and methods are merely exemplary and are in no waylimiting.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of asterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

In addition, the composition can comprise additional therapeutic orbiologically-active agents. For example, therapeutic factors useful inthe treatment of a particular indication can be present. Factors thatcontrol inflammation, such as ibuprofen or steroids, can be part of thecomposition to reduce swelling and inflammation associated with in vivoadministration of the vector and physiological distress. Immune systemsuppressors can be administered with the composition method to reduceany immune response to the vector itself or associated with a disorder.Alternatively, immune enhancers can be included in the composition toupregulate the body's natural defenses against disease.

Antibiotics, i.e., microbicides and fungicides, can be present to reducethe risk of infection associated with gene transfer procedures and otherdisorders.

The invention also provides a method of treating spinal cord injury painor peripheral neuropathic pain in a mammal comprising administering to amammal a vector or composition of the present invention comprising anucleotide sequence encoding a glutamic acid decarboxylase (GAD) proteinin an amount effective to treat spinal cord injury pain or peripheralneuropathic pain. In a preferred embodiment, the administered vector isa viral vector. In a preferred embodiment, the mammal is a human.

The method of treating spinal cord injury pain or peripheral neuropathicpain further can comprise the administration (i.e., pre-administration,co-administration, and/or post-administration) of other treatmentsand/or agents to modify (e.g., enhance) the effectiveness of the method.The method of the invention can further comprise the administration ofother substances which locally or systemically alter (i.e., diminish orenhance) the effect of the composition on a host. For example,substances that diminish any systemic effect of the protein producedthrough expression of the nucleic acid sequence of the vector in a hostcan be used to control the level of systemic toxicity in the host.Likewise, substances that enhance the local effect of the proteinproduced through expression of the nucleic acid sequence of the vectorin a host can be used to reduce the level of the protein required toproduce a prophylactic or therapeutic effect in the host. Suchsubstances include antagonists, for example, soluble receptors orantibodies directed against the protein produced through expression ofthe nucleic acid sequence of the vector, and agonists of the protein.

One skilled in the art will appreciate that suitable methods ofadministering the inventive vector and composition of the invention toan animal (especially a human) for therapeutic or prophylactic purposes,e.g., gene therapy, vaccination, and the like (see, for example,Rosenfeld et al., Science, 252, 431-434 (1991), Jaffe et al., Clin.Res., 39(2), 302A (1991), Rosenfeld et al., Clin. Res., 39(2), 311A(1991), Berkner, BioTechniques, 6, 616-629 (1988)), are available, and,although more than one route can be used to administer the composition,a particular route can provide a more immediate and more effectivereaction than another route. A preferred route of administrationinvolves transduction of DRG neurons through peripheral inoculation torelease GABA in the dorsal horn. In many embodiments, this can beaccomplished by delivering the GAD vector by subcutaneous inoculation,which is an attractive feature of the inventive approach to treat SCIpain or peripheral neuropathic pain.

The dose administered to an animal, particularly a human, in the contextof the invention will vary with the particular vector, the compositioncontaining the vector and the carrier therefor (as discussed above), themethod of administration, and the particular site and organism beingtreated. The dose should be sufficient to effect a desirable response,e.g., therapeutic or prophylactic response, within a desirable timeframe. Thus, the dose of the vector of the inventive compositiontypically will be about 1×10⁵ or more particle units (e.g., about 1×10⁶or more particle units, about 1×10⁷ or more particle units, 1×10⁸ ormore particle units, 1×10⁹ or more particle units, 1×10¹⁰ or moreparticle units, 1×10¹¹ or more particle units, or about 1×10¹² or moreparticle units). The dose of the vector typically will not be 1×10¹³ orless particle units (e.g., 4×10¹² or less particle units, 1×10¹² or lessparticle units, 1×10¹¹ or less particle units, or even 1×10¹⁰ or lessparticle units).

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope. In theseexamples, several measurements were recorded and statistically analyzed.The statistical significance of the difference between vector treatedand control animals was determined using multivariate analysis ofvariance or the Kruskal-Wallis test for non-parametric measures. Singlecomparison was performed with Student's t test, using a P value of <0.05as significant. All data are expressed as means±the standard error ofthe mean (SEM).

Example 1

This example demonstrates the construction of the GAD vector.

The nonreplicating HSV vector QHGAD67 is defective in expression of theHSV immediate early (IE) genes ICP4, ICP22, ICP27, and ICP47, andcontains the human GAD67 gene under the control of the humancytomegalovirus immediate early promoter (HCMV IEp) in the UL41 locus(FIG. 1). Control vector Q0ZHG (constructed according to the methoddescribed in Chen et al. J. Virol, 74(21), 10132-41 (2000)) is defectivein the same genes, but contains the Escherichia coli lacZ reporter genein the same position (FIG. 1).

GAD67 cDNA (constructed according to the method described in Bu, D. F.,et al., Proc. Natl. Acad. Sci. USA, 89, 2115-2119 (1992)) wasindividually sub-cloned as a ClaI/XbaI fragment downstream of the humancytomegalovirus immediate early promoter in the shuttle plasmid p41H,containing the promoter and adequate HSV flanking DNA sequence in orderto enable efficient homologous recombination at the U_(L)41 gene locusof the vector. The expression/targeting cassette was recombined into theU_(L)41 locus of vector Q0ZHG by cotransfection of complementing 7bcells with PmeI-digested viral and targeting plasmid DNA to replace theLacZ marker gene with the GAD expression construct. The recombinantQHGAD67 was purified by three rounds of limiting dilution purificationand the genetic structure confirmed by Southern blot. Vector stocks wereproduced in 7b cells in roller bottles, purified on a continuousnycodenze gradient, and aliquotted and stored at −80° C. until thawedfor use. The titer of the final vector product was determined asdescribed in Krisky, D., et al., In Methods in Molecular Medicine, HumanPress, Totowa, N.J. (1996))

Example 2

This example demonstrates the construction of an HSV vector havingextended deletions of the ICP4 and ICP27 loci and a deletion of UL55.

The schematic for constructing this vector is set forth in FIG. 14.Specifically, plasmid d106 (Hadjipanayis and DeLuca, Can Res 65(12):5310-6 (2005)) was virally crossed with plasmid TOZ.1 (Arafat et al.,Clin Can Res 6: 4442-8 (2000)) to produce QOZHG.1. The details of vectorQOZHG.1 are described in Example 1 and its construction described inChen et al., J Virol 74(21), 10132-41 (2000).

Plasmid pPXE (Niranjan et al., Mol Ther 8(4).530-42 (2003)) wasrecombined into the ICP27 locus of QOZHG.1 to rescue the previous ICP27deletion and to remove HCMV-eGFP gene. A single recombinant was thenisolated, purified and verified by selecting a plaque that did notexhibit green fluorescence under a fluorescent microscope. Therecombinant was termed E1. E1 was negative for the GFP gene and positivefor the LacZ gene.

Plasmid 41HN was produced by cloning the Hind III to Not I fragment(HSV-1 genomic nucleotides 90145 to 93858) containing the UL41 codingsequence into the Hind III to Not I sites of pBSSK (Stratagene). Plasmid41HN was then recombined into the UL41 locus of E1 to rescue thewildtype UL41 gene and remove LacZ. The resulting vector, named E1-1 wasisolated, purified, and verified by standard methods. This vector wasnegative for both gfp and lacz genes.

Plasmid pSASB3 was constructed by cloning the Sph I to Afl III (Sal ITinkered) fragment (1928 bp) of the HSV-1 KOS strain genome (nucleotides124485-126413) into Sph I/Sal I digested pSP72 followed by insertion ofa the 695 bp BglII to BamH I fragment (nucleotides 131931 to 132626)containing regions upstream of the ICP4 promoter including the viralorigin contained within the short inverted repeat regions into the BglII to BamH I sites of the vector plasmid.

Plasmid pSASB3gfp was contructed by cloning a HCMV-eGFP fragment in theBamHI site of pSASB3. Plasmid pSASB3gfp was then recombined into theICP4 locus of E1-1 to expand the ICP4 deletion. The resulting vector,named E1G6/d106-4HG was then isolated, purified, and verified bystandard methods.

The plasmid pSASB3-HPPE was created by cloning a EcoR I to Sal Ifragment of the plasmid pCMV-hPPE from Dr. Steven Wilson, University ofSouth Carolina (Liu F, Housley P R, Wilson S P. [J. Neurochem. 1996October; 67(4):1457-62) containing the HCMV immediate early promoter,SV40 intron (180 bp XhoI-PstI) from the 16s/19s RNA of SV40, the entirehPPE coding sequence, and the SV40 polyadenylation signal (SV40 bases2533 to 2729 into plasmid pSASB3 at the unique Sal I site. Sal I releaseof the hPPE expression construct was made possible by previous EcoR Idigestion followed by Klenow fragment blunting of the EcoR I site andligation with a Sal I linker. The product of this ligation was thendigested with Sal I to purify the Sal I flanked expression construct.pCMV-HPPE was subcloned from a cDNA clone pUR292 from Dr. Barbara Spruceas 946 bp BamHI-HindIII fragment from pUR292 that was blunt-ended, NotIlinkers added, and cloned into the unique NotI site of the expressionplasmid pCMV13.

The final enkephalin expression/ICP4 targeting construct (pSASB3-HPPE)contains the following elements; 1) bases 131931 to 132626 of the HSVgenome to provide a 5′ recombination flanking sequence targeting theICP4 locus, 2) the human cytomegalovirus (HCMV) immediate early promoter(IEp), 3) the SV40 16s/19s intron splice donor and acceptor sites, 4)the preproenkephalin cDNA, 5) the SV40 late polyadenylation signal, 6)bases 124485 to 126413 of the HSV genome to provide a 3′ recombinationflank targeting the ICP4 locus.

Plasmid pSASB3-hppe was then recombined into the ICP4 locus ofE1G6/d106-4HG to produce the NurelP1 (NPP1) vector, which is genericallyknown as 6221.

Plasmid PS-UB6R-6 was created by cloning a Bgl II-BamH I flankedubiquitin promoter driven Red2 (Invitrogen) in the BamH I site of PSP4.The BamH I site of PSP.4 is located in between the 5′ ICP27 flankfragment and the UL56 coding sequence.

Plasmid PS-UB6R-6 was recombined into the ICP27 locus of NP1 to expandthe ICP27 deletion to include all of ICP27 and UL55 and to insert theUB6-Red gene. The resulting vector, termed HPPE6221R, was isolated,purified, and verified by selecting red plaques.

Plasmid PSP4 was created by cloning the EcoR I to BamH I (HSV-1 genomic110095 to 113322) sequence 5′ to the ICP27 coding sequence into theplasmid PS.2. PS.2 contains the Dde I to Sma I (HSV-1 genomic fragment116156 to 117119) blunt end ligated into the Not I site of pBSSK(Stratagene).

Plasmid PSP4 was recombined into the ICP27 locus of HPPE6221R vector toremove UB6-Red and to leave the ICP27/UL55 deletion. The resultingvector, termed NurelP2 (NP2), was isolated, purified, and verified bystandard methods.

Plasmid pSASB3GFP was recombined into the ICP4 locus of NP2 to replaceHCMV-hppe with HCMV-eGFP. The resulting vector, termed SAS2, was thenisolated, purified, and verified by standard methods.

Plasmid pRC2 (Invitrogen) was modified sequentially by converting the 1)HinDIII site into a ClaI site, 2) the BbvII site into a HinDIII site, 3)the resulting HinDIII site into a BglII site to make plasmid pRC2HB2.The BglII fragment of pRC2HB2, about 1200 base pairs, was cloned intothe BamHI site of plasmid pSASB3 creating plasmid pSHB3. Separately theGAD67 clone was modified by converting the HinDIII site into a BamHIsite creating pGADHB2. The resulting 2.8 kb BamHI fragment from pGADHB2was cloned into the BamHI site of pSHB3 to make pGADL1.

Plasmid pGAD-L1 was recombined into the ICP4 locus of SAS2 to producethe vector NurelG2 (NG2).

The vectors NP2, SAS2 and NG2 are vectors that have no homology with thecell line, such that no homologous recombination can take place betweenthe cell line and the vector. Therefore, these vectors are ideal forusing in combination with the ICP4, ICP27, UL55 complementing cell linefor vector production.

Example 3

This example demonstrates the expression of GAD protein by GAD vectortransduced cells.

One week after subcutaneous inoculation of QHGAD67 into the plantarsurface of the hind paw of a laboratory rat the amount of GAD67 mRNA inthe pooled L4-L6 DRG detected by real-time RT-PCR was fivefold greaterthan in contralateral DRG transduced with Q0ZHG (FIG. 2). Also, GAD67immunoreactivity in transduced DRG was present in neurons in a broadspectrum of DRG neurons of all sizes compared to the contralateral(vehicle-injected) DRG. GAD67 protein, determined by Western blot, wassignificantly increased in both the lumbar DRG (0.048±0.009 OD units)compared to sham-inoculated controls (0.025±0.006 OD units, P<0.01.

One week after subcutaneous inoculation of 30 μl of 11×10⁹ pfu/mlQHGAD67 increased immunoreactivity was seen predominantly in laminae IIand III compared to the contralateral (vehicle-injected) dorsal horn. Inthe superficial dorsal horn of control rats GAD67 immunoreactivity waslocated predominantly in small round cells of lamina III with fewneuritic extensions that appeared to be endogenous GABA-ergicinterneurons in the superficial dorsal horn and some smaller, punctuate,densely staining, irregular profiles that appeared to be varicosities orsmall axonal terminals. The intensity of immunostaining was increasedsubstantially in the dorsal horn containing central terminals of theaxons from DRG transduced with QHGAD67 and the increasedimmunoreactivity appeared to be located in the irregular profilesrepresenting axonal terminals. By Western blot the amount of GAD in thedorsal spinal cord of the lumbar segments containing the centralterminal of those axons (0.041±0.008 OD units) was significantlyincreased compared to the sham-inoculated controls (0.027±0.004 ODunits) (P<0.01, FIG. 3).

The analysis of GAD RNA by real-time RT-PCR was performed as follows:L4-6 DRGs were rapidly removed and total RNA extracted from the pooledL4-6 ganglia using TriReagent (Sigma). After DNase I digestion,first-strand cDNA was produced using Omniscript reverse transcriptase(Qiagen, Valencia, Calif., USA). Primers and probes for GAD67 and GAPDH(Synthegen, Houston, Tex., USA) were designed using Primer Express(Applied Biosystems, Foster City, Calif., USA). GAD67 forward primer SEQID NO: 3; reverse primer SEQ ID NO: 4; probe, SEQ ID NO: 5 (Synthegen);and GAPDH forward primer SEQ ID NO: 6; reverse primer SEQ ID NO: 7;probe SEQ ID NO: 8 (Invitrogen). PCRs were performed in an ABI Prism7700 sequence detection system (Applied Biosystems) in a total volume of50 μl. The amount of RNA was determined using GAPDH as an internalcontrol and calculated relative to DRG transduced with Q0ZHG. Each PCRamplification was performed in triplicate wells, using the followingconditions: 2 minutes at 50° C. and 10 minutes at 95° C., followed by 40cycles of 15 s at 95° C. and 1 minute at 60° C.

The amount of GAD67 protein was determined by Western blot according tothe following protocol. The L4-L6 DRG or the dorsal quadrant of thelumbar enlargement (L4 to L6 segments) of spinal cord was sonicated inhomogenization buffer (100 mg tissue/ml) consisting of 60 mM phosphatebuffer, pH 7.4, 1 mM phenylmethylsulfonyl fluoride and 0.5% TritonX-100.The homogenate was centrifuged for 15 minutes at 100,000 g, using aTL100 ultracentrifuge (Beckman), and the total protein in thesupernatant was measured by Bradford assay (BioRad, Hercules, Calif.,USA). Proteins were separated on a 4-15% SDS gradient polyacrylamidegel, transferred to nitrocellulose membrane (Immobilon-P, Millipore,Billerica, Mass., USA), incubated with rabbit anti-GAD67 (1:4000,Chemicon, Temecula, Calif., USA) followed by horseradishperoxidase-conjugated goat anti-mouse (1:10,000, Jackson Laboratories,Bar Harbor, Me., USA), and detected by enhanced chemiluminescence (NEN,Boston, Mass., USA). The membranes were stripped and reprobed withrabbit anti-β-actin as a loading control. The intensity of each band wasdetermined by quantitative densitometry using a PC-based image analysissystem (MCID, Imaging Research, Brock, Ontario, Canada).

Example 4

This example demonstrates the increased release of GABA in GAD vectortransduced cells.

Primary DRG neurons transduced in vitro with QHGAD67 at a multiplicityof infection (m.o.i.) of 1 released GABA into the medium (9.53±2.15pmol/10 μl) in amounts substantially greater than those released fromcontrol (2.34±0.22 pmmol/10 μl, P<0.01) or Q0ZHG-transduced DRG neurons(2.56±0.54 μmol/10 μl, P<0.01) (FIG. 4A). The in vivo amount of GABAreleased into the dorsal spinal cord from the central terminals of DRGtransduced by subcutaneous inoculation into the foot 1 week earlier wasdetermined by microdialysis from a catheter implanted in the ipsilaterallumbar dorsal horn. GABA contained in the dialysate collected fromanimals inoculated with Q0ZHG contained 0.74±0.24 pmol/10 μl compared tothe dialysate from animals inoculated with QHGAD67 which contained1.46±0.25 pmol/10 μl (P<0.05) (FIG. 4B).

Dissociated DRG neurons from 17-day-old rat embryos were plated onpoly-D-lysine-treated coverslips at a density of 10⁵ cells/well in a24-well plate. Each well contained 500 μl Neurobasal medium containingB27, Glutamax I, Albumax II, and penicillin/streptomycin (Gibco-BRL,Carlsbad, Calif.), supplemented with 100 ng/ml of 7.0S NGF (Sigma, St.Louis, Mo.). At 14 days in culture, the cells were infected with eitherQHGAD67 or Q0ZHG at a m.o.i. of 1 for 1 hour, after which the virus wasremoved. Forty-eight hours later the medium was changed to 100 μl ofartificial cerebrospinal fluid and after 5 minutes collection time thebathing solution was centrifuged 5 minutes at 10,000 g and thesupernatant taken for determination of GABA by HPLC. The DRG cells wereexamined for expression of GAD67 protein by immunocytochemistry.

The amount of GABA released from nerve terminals in the dorsal horn invivo was determined by HPLC of a microdialysate using the followingprotocol: Rats were anesthetized with chloral hydrate (400 mg/kg), thelaminae of T11 and T12 vertebrae that overlie the lumbar segments of thespinal cord were removed, leaving the dura intact, and the animals werefixed in a stereotaxic apparatus. A heating lamp was used to preventheat loss and the body temperature was kept at 37.5° C. using a feedbacksensor. A small dural incision lateral to midline was made with a sharpneedle, and a microdialysis probe (CMA/11, cuprophane dialysis membrane,length 1 mm, diameter 0.24 mm, molecular cut-off 6 kDa,CMA/Microdialysis, Stockholm, Sweden) was inserted into the dorsal hornthrough the dural incision and perfused with artificial cerebrospinalfluid (CMA/Microdialysis) at a rate of 1 μl/min. After 1 h to allow forequilibration with the extracellular fluid, samples were collected for 1h. At the end of the experiment, the probe was checked for the presenceof air bubbles and the position in the dorsal horn of the spinal cordverified by microscopy of perfusion-fixed sections.

Next, the amount of GABA released from transduced cells in vitro, orcollected by microdialysis in vivo, was determined using HPLC with6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization(AccQ.Fluor Reagent Kit; Waters, USA). Twenty microliters of culturesolution was mixed with 20 μl of derivatizing reagent in 60 μl of boratebuffer; 10 μl of the sample was allowed to react for 10 minutes at 55°C. and separated by gradient HPLC (Waters 2695 Separations Module) on anAccQ.Taq column (3.9×150 mm; Waters) with a mobile phase consisting ofFluent A (Waters) and acetonitrile and a flow rate of 1 ml/min. Thepeaks were detected by fluorescence at 37° C. (Waters 2475 Detector)using an excitation wavelength of 250 nm and emission wavelength of 395nm.

The intrathecal catheter was surgically implanted in the laboratory rataccording to the following protocol: An intrathecal catheter was placed1 week after T13 left spinal hemisection using a modification of themethod of Storkson. Briefly, the animals were reanesthetized withchloral hydrate (400 mg/kg), a longitudinal incision was made from L2 toL6 a few millimeters left of the midline, and a polyethylene catheter(PE-10, Clay Adams, Parsippany, N.J., USA) was introduced from the L4-L5intervertebral space into the lumbar subarachnoid space so that the tipof the catheter was located near the lumbar enlargement of the spinalcord. The distal end of the catheter was tunneled subcutaneously toemerge at the neck, leaving 7 μl of dead space. After implantation ofthe intrathecal catheter, the rats were housed in individual cages andthose animals showing evidence of motor dysfunction were sacrificed. Thelocation of the catheter tip was confirmed by infusion of 15 μl oflidocaine (20 mg/ml) followed by 8 μl of saline to produce a motorparalysis lasting for 20-30 minutes. Intrathecal drug administration wasperformed using a microinjection syringe (Hamilton Co., Reno, Nev., USA)connected to the intrathecal catheter in awake, briefly restrained rats.

Example 5

This example demonstrates that subcutaneous inoculation of a GAD vectorin a laboratory rat reduces mechanical allodynia and thermalhyperalgesia after SCI.

One day after T13 left spinal cord hemisection, all animals showedipsilateral hind-limb paralysis with no motor dysfunction in the hindlimb contralateral to spinal cord hemisection. Two weeks after spinalcord hemisection, there was considerable return of motor function (BBBscore of 12-13, data not shown). A BBB score of 12, corresponding tofrequent-to-consistent weight-supporting phantom steps and occasionalfront leg-hind leg coordination is sufficient to allow full behavioraltesting of somatosensory-induced paw withdrawal. At one week afterspinal cord hemisection, mechanical allodynia, manifested by asignificant decrease in hind-paw withdrawal threshold to a graded seriesof von Frey filament stimuli (1.71±0.35 g ipsilateral, 1.9±0.52 gcontralateral) as compared to the preoperative threshold (11.2±1.68 gipsilateral, 11.6±1.71 g contralateral), in both hind paws was observed.Inoculation of QHGAD67 (1×10⁹ pfu/ml, 30 μl/paw) subcutaneously in theplantar surface of the hind paws bilaterally one week after spinal cordhemisection significantly increased the hind-paw withdrawal threshold(4.4±1.12 g ipsilateral, 4.1±0.75 g contralateral) measured one weekafter inoculation (two weeks after injury). Control animals inoculatedwith Q0ZHG showed no change in their mechanical threshold (1.8±0.28 gipsilateral, 1.6±0.34 g contralateral, P<0.01 compared to QHGAD67). Themaximal antiallodynic effect (i.e., increase in paw withdrawalthreshold) occurred two weeks after QHGAD67 inoculation (5.19±0.82 gipsilateral and 5.8±1.14 g contralateral) and the antiallodynic effectpersisted for five weeks, decreasing to 2.86±0.63 g ipsilateral and3.2±0.47 g contralateral at six weeks after inoculation (FIGS. 5A and5B). Reinoculation with the same dose of QHGAD67 into the footpadsbilaterally at six weeks after initial inoculation reestablished theantiallodynic effect. The magnitude of the effect obtained byreinoculation was at least as great as that produced by the initialinjection of the vector, and the duration of the effect produced byreinoculation was slightly longer (6-7 weeks) than that which resultedfrom the initial inoculation. There was no significant difference in pawwithdrawal thresholds between Q0ZHG-inoculated and vehicle-treated ratsat all time points (FIGS. 5A and 5B).

After spinal cord hemisection, animals also demonstrated thermalhyperalgesia manifested by a decrease in withdrawal latency in responseto noxious thermal stimuli (6.7±0.51 s ipsilateral, 6.9±0.6 scontralateral) compared to the preoperative values (12.6±1.23 sipsilateral, 12.1±1.25 s contralateral). One week after inoculation ofQHGAD67 (two weeks after spinal cord hemisection) there was astatistically significant increase in thermal latency (8.8±0.48 sipsilateral, 8.7±0.71 s contralateral) compared to Q0ZHG-inoculatedcontrols (6.5±0.43 s ipsilateral, 7.1±0.42 sec contralateral). The timecourses of antihyperalgesic effect were similar to those ofantiallodynic effect of the vector (FIGS. 5C and 5D), except that thepeak effect occurred four weeks after inoculation (9.7±0.71 sipsilateral, 9.62±0.78 s contralateral). Reinoculation of QHGAD67 vectorat six weeks also reestablished the antihyperalgesic effect. Theduration and the magnitude of the antihyperalgesic effect after thesecond inoculation was longer and greater than those which followed theinitial inoculation. There was no significant difference betweenvehicle-treated and Q0ZHG-inoculated animals in both hind-paw withdrawallatencies at any time period (FIGS. 5C and 5D).

For these tests, male Sprague-Dawley rats, weighing 175-200 g were used.Housing conditions and experimental procedures were approved by theUniversity of Pittsburgh Institutional Animal Care and Use Committee.With the rat under chloral hydrate anesthesia (400 mg/kg) the T11-T12spinal laminae were located by palpating the last rib (attached to T13).A longitudinal incision was made exposing several segments, and alaminectomy was performed at two vertebral segments (T11-T 12). Thelumbar enlargement was identified by accompanying dorsal vessels, andthe spinal cord was hemisected at T13 using a No. 11 scalpel blade withcare taken not to damage the major dorsal vessels and vascular branches.A tuberculin syringe with a 28-gauge needle was placed dorsoventrally atthe midline of the cord and pulled laterally to ensure that the spinalcord hemisection was complete. Muscle and fascia were sutured closed,and the skin was closed with autoclips. Following surgery, animals weremaintained under the same preoperative conditions. All the animals wereeating and drinking within 3 hours after surgery. Locomotor function wasobserved and recorded using the BBB Locomotor Rating Scale to ensurethat motor recovery of the limb ipsilateral to the spinal cordhemisection was sufficient to allow for somatosensory behavioraltesting. Animals that demonstrated a loss of locomotion in both hindlimbs, indicating bilateral corticospinal tract transaction, wereexcluded from the study at that time. Animals that met the criteria werethen inoculated with a vector. Six animals in each group were inoculatedwith a vector and tested by reinoculation.

Behavioral testing for mechanical allodynia and thermal hyperalgesia wasperformed during the day portion of the circadian cycle (8:00 AM to 5:00PM). Mechanical allodynia was assessed by measuring the threshold ofpaw-withdrawal response to graded mechanical stimuli using a series ofvon Frey filaments (0.4, 0.7, 1.2, 1.5, 2.0, 3.6, 5.5, 8.5, 11.8, and15.1 g). Rats were placed in transparent plastic cubicles on a meshfloor for a period of at least 30 minutes for acclimatization, afterwhich von Frey filaments were applied to the plantar surface of the footserially in ascending order of strength with sufficient force to causeslight bending against the paw and held for 6 s. A brisk foot withdrawalto von Frey filament application was regarded as a positive response andcause to present the next weaker stimulus. Thermal hyperalgesia wasassessed by measuring the latency of paw withdrawal from a radiant heatsource. In this test, the rats were placed on a glass plate over a lightbox. After a ten minute habituation period the plantar surface of thepaw was exposed to a beam of radiant heat applied through the glassfloor. The light beam was turned off automatically by a photocell whenthe rat lifted the limb, allowing the measurement of time between thestart of the light beam and the paw withdrawal. This time was defined asthe paw withdrawal time. Testing was always performed in triplicate atfive minute intervals and twenty seconds was used as the cut-off time.

Example 6

This example demonstrates that the behavioral effects of GAD vectorinoculation are reversed by bicuculline and phaclofen.

The pharmacologic basis of the QHGAD67-mediated antinociceptive effectusing the GABA_(A) receptor-selective antagonist bicuculline and theGABA_(B) receptor-selective antagonist phaclofen was examined.Intrathecal administration of bicuculline (0.5 μg; Sigma) or phaclofen(0.8 μg; Sigma) to sham-operated animals two weeks after surgery did notalter the mechanical threshold or thermal latency. Administration of thesame dose of bicuculline to rats with spinal cord hemisection inoculatedwith QHGAD67 reduced the mechanical threshold from 4.87±1.13 g to3.5±0.7 g (P<0.05) ipsilateral to the spinal cord hemisection and from5.75±1.41 g to 3.38±0.9 g (P<0.01) contralateral to the spinal cordhemisection (FIG. 6A) measured 10-15 minutes after drug administration.Intrathecal phaclofen reduced the mechanical threshold to 3.6±0.78 g(P<0.05) ipsilateral to the spinal cord hemisection and 4.05±0.75 g(P<0.05) contralateral to the spinal cord hemisection (FIG. 6A). Thethermal withdrawal latency was reduced from 9.28±1.39 s to 7.23±1.21 s(P<0.05) ipsilateral, and from 9.56±1.5 s to 7.41±1.29 s P<0.05)contralateral by administration of bicuculline and to 7.54±1.16 s(P<0.05) ipsilateral, 7.66±1.24 s (P<0.05) contralateral byadministration of phaclofen (FIG. 6B). In each case the effect of thedrug was measured at the peak effect 30 minutes after drugadministration. One hour after inoculation there was no longer anydetectable drug effect. There were no significant changes in eithermechanical threshold or thermal latency in spinal cord hemisected ratsinoculated with Q0ZHG and treated with bicuculline or phaclofen at thesame doses.

Example 7

This example demonstrates that transduction of cells with a GAD vectorreduces CGRP immunoreactivity in the dorsal horn.

In sham-operated animals, CGRP immunoreactivity at the L5 segment wasweak, confined largely to laminae I and II within the superficial dorsalhorn of the spinal cord bilaterally. One week after left T13hemisection, CGRP immunoreactivity was increased and staining could bedetected extending into laminae III and IV of dorsal spinal cordbilaterally. In spinal cord hemisected rats 1 week after inoculation ofQHGAD67 in both hindpaws, CGRP-like immunoreactivity was reduced(76.5±13.3 OD unit ipsilateral, 63.6±12.4 OD unit contralateral)compared to spinal cord hemisected rats inoculated with Q0ZHG(107.3±22.4 OD unit ipsilateral, 86±23.5 unit contralateral, P<0.0-5) orvehicle-treated animals (96.7±21.8 OD unit ipsilateral, 104.6±22.6 ODunit contralateral, P<0.05). There was no significant difference instaining between Q0ZHG-inoculated and vehicle-treated animals (FIG. 7).

The distribution of GAD protein in unlesioned transduced animals andCGRP peptide in lesioned transduced animals was determined byimmunohistochemistry. Rats were perfused intracardially with 4%paraformaldehyde in 0.1 M phosphate buffer, the L5 segment of spinalcord and attached roots removed, postfixed in the same solution for twohours, and cryoprotected with 30% sucrose in PBS for two days.Twenty-micrometer cryostat sections were thaw mounted onto coldSuperfrost microscope slides (Fisher, Pittsburgh, Pa., USA) andincubated overnight at 4° C. with rabbit anti-GAD67 (1:2000, Chemicon)or rabbit anti-CGRP (1:500, PLI, San Carlos, Calif., USA) followed byfluorescent anti-rabbit IgG (Alexa Fluor 594, 1:500, Molecular Probes,Eugene, Oreg., USA) for two hours at room temperature. Fluorescentimages were captured by confocal microscopy (Diagnostic Instruments,Sterling Heights, Mich., USA).

Example 8

This example demonstrates that transfer of the gene encoding GAD todorsal root ganglion using a herpes simplex virus vector attenuatesperipheral neuropathic pain.

Male Sprague-Dawley rats weighing 225 to 250 gm underwent selective L5SNL (as described in Hao et al., Pain; 102:135-42 (2003)). One weekafter SNL, 30 μl of vector (either QHGAD67 or Q0ZHG, 4×10⁸plaque-forming units per milliliter) was injected subcutaneously in theplantar surface of the left hind paw, ipsilateral to the ligation.Mechanical allodynia induced by SNL was determined by assessing theresponse of paw withdrawal to von Frey hairs of graded tensile strength(see Hao et al., supra, and Chaplan et al., J Neurosci Methods, 53:55-63(1994)) with a tactile stimulus producing a 50% likelihood of withdrawaldetermined using the up-down method (see Dixon et al., Annu RevPharmacol Toxicol; 20:441-62 (1980)) Thermal hyperalgesia was determinedusing a Hargreaves apparatus (as described in Hargreaves et al., Pain;32:77-88 (1988)) recording the time to withdrawal from a radiant thermalstimulus positioned directly under the hind paw.

After L5 SNL, rats displayed a significant decrease in the magnitude ofthe mechanical stimulus necessary to evoke a brisk withdrawal responseto von Frey hair stimulation (FIG. 11A) and a significant reduction inlatency to withdraw from a heat stimulus (thermal hyperalgesia; see FIG.11B). Rats inoculated with QH-GAD67 showed a statistically significantincrease in mechanical threshold beginning 1 week after inoculation. Theantiallodynic effect of QHGAD67-mediated GABA expression was sustainedand continuous, lasting 5 to 6 weeks and peaking at 2 weeks afterinoculation (see FIG. 11A). The peak value of mechanical threshold, 8.6gm, was close to the preoperative value. By 7 weeks after inoculation,the antiallodynic effect of vector transduction disappeared, and themechanical threshold of QHGAD67-injected rats was identical to that ofcontrol rats. Reinoculation into the same paw with the same dose ofQHGAD67 reestablished the antiallodynic effect (see FIG. 11A). SNLinduced a decrease in the thermal latency from 10.7 to 6.5 seconds,which lasted 3 weeks before gradually recovery. Rats inoculated withQHGAD67 showed a statistically significant increase in thermal latencyin the ipsilateral paw beginning 1 week after inoculation (see FIG.11B), an effect that was sustained and continuous, lasting 3 to 4 weeks(see FIG. 11B). Sham-operated animals had no change in mechanicalthresh-old or thermal latency.

Expression of c-Fos and phosphorylated extracellular signal-regulatedkinase 1 and 2 (p-ERK1/2) induced by gentle touch is one indirectbiological marker of nociceptive processes (Catheline et al., Pain;92:389-98 (2001)). Three weeks after SNL, gentle touch was applied onceevery 4 seconds for 10 minutes, with the flat surface of theexperimenter's thumb to the rat's paw, and the number of immunoreactivecells (anti-c-Fos or anti-p-ERK1/2 antibodies; Santa Cruz Biotechnology,Santa Cruz, Calif.) detected avidin-biotin horseradish peroxidasefollowed by nickel-enhanced diaminobenzidine (Vector Laboratories,Bur-lingame, Calif.). 12 The number of Fos-LI-positive neurons wassubstantially increased ipsilateral to SNL compared with sham-operatedcontrol rats, and inoculation of vector QHGAD67 significantly reducedthe number Fos-LI positive neurons in laminae I-VI (see FIG. 12).p-ERK1/2 expression in laminae I and II was also increased in rats aftergentle touch stimulation with SNL, and that increase was blocked inanimals inoculated with QHGAD67. p-ERK was not induced by 10 minutes ofgentle tactile stimulation in sham-operated animals but it wassubstantially induced after spinal nerve ligation (SNL) in animalsinoculated with QOZHG. Touch-induced expression of p-ERK1/2 wassuppressed in animals inoculated with QHGAD67, confirmed by counts ofp-ERK1/2-positive neurons in the dorsal horn (FIG. 13).

These results demonstrate that subcutaneous inoculation of an HSV vectorexpressing GAD to transduce DRG in vivo attenuated the behavioralmanifestations of mechanical allodynia and thermal hyperalgesia in amodel of peripheral neuropathic pain; the effect on behavior wasconfirmed by histological measures showing a block in the induction ofexpression of c-Fos and p-ERK1/2 in the ipsilateral spinal dorsal horn.

DISCUSSION

The lateral hemisection of a laboratory rat's spinal cord at T13produces bilateral SCI pain related behavior below the lesion in bothhind limbs (“the SCI model”). The SCI pain related behavior ismanifested as mechanical allodynia and thermal hyperalgesia. Thisphenomenon is accompanied by bilateral spinal cord reorganization. Inthe above mentioned experiments, this SCI model was used to examine theeffects of the local production and release of GABA due to a GAD vectormediated gene transfer to the lumbar DRG in alleviating some of themanifestations of SCI pain.

DRG neurons were transduced with a HSV vector encoding for GAD (“the GADvector”). These transduced DRG neurons expressed GAD in vitro and invivo. The expression of GAD in these cells resulted in the release ofGABA. In the laboratory rats subjected to lateral hemisection of thespinal cord at T13 followed by subcutaneous inoculation with the GADvector, regional GABA release from transduced DRG neurons reducedmechanical allodynia and thermal hyperalgesia in the hind limbs. Thiseffect could be reversed by either GABA_(A) or GABA_(B) receptorantagonists administered at doses that did not alter nociception innormal animals or in laboratory rats subjected to lateral hemisection ofthe spinal cord at T13 without subcutaneous inoculation with the GADvector. Moreover, GAD vector mediated GABA release also attenuated theincrease in CGRP immunoreactivity in the lumbar dorsal horn that occursafter SCI. Therefore, the inventive method involving GAD vector mediatedgene transfer to DRG can effectively be used to treat below levelneuropathic SCI pain.

Release of GABA from primary DRG neurons transduced in vitro with theGAD vector was not increased in a medium containing 60 mM K⁺ and wasunaffected by removal of Ca²⁺ from the medium suggesting that GABArelease is not vesicular, but occurs constitutively, perhaps throughreversal of the GABA transporter. Although the amount of GABA releasedfrom nerve terminals in vivo was sufficient to elevate GABA levels inmicrodialysate of the dorsal horn significantly, there was no evidenceof motor weakness in these animals, suggesting that transgene mediatedGABA release was limited to the dorsal horn. This would be consistentwith the observation that animals transduced by subcutaneous inoculationof the proenkephalin expressing HSV vector acquired an analgesic effectthat was limited to the limb ipsilateral to the inoculation.

In laboratory rats with uninjured spinal cords, tonic GABA-ergicinhibition of low threshold afferent inputs modulates sensoryprocessing, and bicuculline block of GABA receptor function producespain-related behavior. Electrophysiologic studies suggest that bothGABA_(A) and GABA_(B) receptors contribute to the tonic modulation ofnociceptive neurotransmission at the spinal cord level. Peripheral nerveinjury results in a decrease in GABA levels in the dorsal horn and areduction in the primary afferent evoked inhibitory post synapticcurrents in the dorsal horn after partial nerve injury. However, whetherthese phenomena result from a loss of GABA-ergic interneurons or adesensitization of GABA receptors as found in central sensitization hasnot been fully established. A transient reduction in GABAimmunoreactivity has been reported in the lumbar spinal cord afterphotochemical induction of spinal ischemia, but previous studies havenot examined GABA immunoreactivity below the level of hemisection.Nonetheless, constitutive delivery of GABA to the dorsal horn below thelevel of the hemisection as a result of GAD vector transduction of DRGneurons reduced behavioral measures of mechanical allodynia and thermalhyperalgesia after SCI. This indicates that SCI pain is susceptible tomodulation by GABA.

The effect of baclofen on central neurogenic pain has been assessed inseveral trials with generally positive results in brief trials, but withless remarkable long-term relief. Intrathecal administration of baclofenpartially alleviates chronic mechanical and cold allodynia in anischemia model of SCI. In a chronic constriction injury model ofneuropathic pain, intrathecal transplantation of GABA-releasing cellsreverses some of the manifestations of neuropathic pain. It has beendiscovered that the pain-relieving effects of GAD vector mediated GABArelease were continuous over the course of several weeks.

The amount of GABA release over time was not measured. However, it wasobserved that the reinoculation of the GAD vector after the analgesiceffect had waned reestablished the reduction in mechanical allodynia andthermal hyperalgesia. This observation demonstrates that the loss oftherapeutic effect was not due to the development of tolerance, but to areduction in gene expression. This is consistent with the findings ofprevious studies that examined vectors in which transgene expression wasdriven by transiently active human cytomegalovirus immediate earlypromoter (HCMV IEp).

There are no “objective” measures of pain and pain relief. However,measurements of the increase in the amount of CGRP immunoreactivity inspinal cord segments below the level of injury in the SCI model havebeen used to measure pain and pain relief in the laboratory.Unfortunately, little is known about the phenomena of increased spinalcord CGRP in the SCI model. The mechanisms responsible for the increasein below-level CGRP have not been defined. It is not known whether theincrease in immunoreactivity correlates with increased release onturnover of CGRP. It is also not known whether CGRP plays any role inthe SCI pain phenomenon or occurs as an epiphenomenon of SCI.Nonetheless, the increase in CGRP that occurs after SCI in these studiesserves as a histologic correlate to the behavioral measures of painanalogous to the increase in c-fos immunoreactivity induced bynonnoxious touch in the spinal cord nerve ligation model of peripheralneuropathic pain. Therefore, measurements of CGRP can be used todetermine the effect of GAD vectors in treating SCI pain. While notwishing to be bound to any particular theory, because CGRP is located inthe unmyelinated and thinly myelinated afferents that projectprincipally to laminae I, II, and V, of the dorsal horn, the increase inCGRP is believed to result from the sprouting of primary afferents. Themechanism through which GABA release from GAD transduced cells preventsthe increase in CGRP expression is not known.

From the foregoing, in accordance with the invention, a nonreplicatingHSV vector designed to express human glutamic acid decarboxylase (GAD)was successfully constructed, and this vector was used to treat SCI painand also peripheral neuropathic pain. These examples demonstrate thatthe inventive method involving the use of a gene transfer approach canbe used to transduce DRG neurons through peripheral inoculation torelease GABA in the dorsal horn. These examples also demonstrate thatthe inventive method involving gene transfer using the GAD vectorreduces below-level mechanical allodynia and thermal hyperalgesia afterSCI. The ability to deliver the GAD vector by subcutaneous inoculationis an attractive feature of the inventive approach to treat SCI pain andalso peripheral neuropathic pain.

The practice of the present invention employs, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. (See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNACloning: A Practical Approach, Vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition); CRC Handbook of Parvoviruses, Vol. I & II (P. Tijessen, ed.);Fundamental Virology, 2nd Edition, Vol. I & II (B. N. Fields and D. M.Knipe, eds.))

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A cell line complementing herpes simplex virus (HSV) ICP4, ICP27, andUL55 genes.
 2. A combination comprising a cell complementing HSV ICP4,ICP27, and UL55 genes and an HSV vector comprising deletions in ICP4,ICP27, and UL55 genes, wherein the cell contains the complementing ICP4,ICP27, and UL55 genes in a non-overlapping fashion with the HSV vector.3. The combination of claim 2, wherein the vector comprises a transgene.